Autoimmune Disease and Modulation of the Immune Response
Autoimmune disease is any disease caused by immune cells that become misdirected at healthy cells and/or tissues of the body. Autoimmune disease affects 3% of the U.S. population, and likely a similar percentage of the industrialized world population (Jacobson et al., Clin Immunol Immunopathol 84: 223-43, 1997). Autoimmune diseases are characterized by T and B lymphocytes that aberrantly target self-proteins, -polypeptides, -peptides, and/or other self-molecules, causing injury and or malfunction of an organ, tissue, or cell-type within the body (for example, pancreas, brain, thyroid or gastrointestinal tract) to cause the clinical manifestations of the disease (Marrack et al., Nat Med 7: 899-905, 2001). Autoimmune diseases include diseases that affect specific tissues, as well as diseases that can affect multiple tissues. For some diseases, this may, in part, depend on whether the autoimmune responses are directed to an antigen confined to a particular tissue, or to an antigen that is widely distributed in the body. The characteristic feature of tissue-specific autoimmunity is the selective targeting of a single tissue or individual cell type. Nevertheless, certain autoimmune diseases that target ubiquitous self-proteins can also affect specific tissues. For example, in polymyositis, the autoimmune response targets the ubiquitous protein histidyl-tRNA synthetase, yet the clinical manifestations primarily involve autoimmune destruction of muscle.
The immune system employs a highly complex mechanism designed to generate responses to protect mammals against a variety of foreign pathogens, while at the same time preventing responses against self-antigens. In addition to deciding whether to respond (antigen specificity), the immune system must also choose appropriate effector functions to deal with each pathogen (effector specificity). A cell critical in mediating and regulating these effector functions is the CD4+ T cell. Furthermore, it is the elaboration of specific cytokines from CD4+ T cells that appears to be the major mechanism by which T cells mediate their functions. Thus, characterizing the types of cytokines made by CD4+ T cells as well as how their secretion is controlled is extremely important in understanding how the immune response is regulated.
The characterization of cytokine production from long-term mouse CD4+− T cell clones was first published more than 20 years ago (Mosmann et al., J Immunol 136: 2348-2357, 1986). In these studies, it was shown that CD4+ T cells produced two distinct patterns of cytokine production, which were designated T helper 1 (Th1) and T helper 2 (Th2). Th1 cells were found to produce interleukin-2 (IL-2), interferon-γ (IFN-γ) and lymphotoxin (LT), while Th2 clones predominantly produced IL-4, IL-5, IL-6, and IL-13 (Cherwinski et al., J Exp Med 169:1229-1244, 1987). Somewhat later, additional cytokines, IL-9 and IL-10, were isolated from Th2 clones (Van Snick et al., J Exp Med 169:363-368, 1989) (Fiorentino et al., J Exp Med 170:2081-2095, 1989). Finally, additional cytokines, such as IL-3, granulocyte macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α) were found to be secreted by both Th1 and Th2 cells. Recently, it was reported that CD4+ T cells isolated from the inflamed joints of patients with Lyme disease contain a subset of IL-17-producing CD4+ T cells that are distinct from Th1 and Th2 (Infante-Duarte et al., J. Immunol 165:6107-6115, 2000). These IL-17-producing CD4+ T cells are designated Th17. IL-17, a proinflammatory cytokine predominantly produced by activated T cells, enhances T cell priming and stimulates fibroblasts, endothelial cells, macrophages, and epithelial cells to produce multiple proinflammatory mediators, including IL-1, IL-6, TNF-α, NOS-2, metalloproteases, and chemokines, resulting in the induction of inflammation. IL-17 expression is increased in patients with a variety of allergic and autoimmune diseases, such as RA, MS, inflammatory bowel disease (IBD), and asthma, suggesting the contribution of IL-17 to the induction and/or development of such diseases.
There is ample evidence showing that suppressor T cells, now called regulatory T cells (Treg cells), suppress autoreactive T cells as an active mechanism for peripheral immune tolerance. Thus far, it is firmly established that Treg cells can be divided into two different subtypes, namely natural (or constitutive) and inducible (or adaptive) populations according to their origins (Mills, Nat Rev Immunol 4:841-855, 2004). In addition, a variety of Treg cell subsets have been identified according to their surface markers or cytokine products, such as CD4+ Treg cells (including natural CD4+CD25+ Treg cells, IL-10-producting Tr1 cells, and TGF-β-producing Th3 cells), CD8+ Treg cells, Veto CD8+ cells, γδ T cells, NKT (NK1.1+CD4−CD8−) cells, NK1.1−CD4−CD8− cells, etc. Accumulating evidence has shown that naturally occurring CD4+CD25+ Treg cells play an active role in down-regulating pathogenic autoimmune responses and in maintaining immune homeostasis (Akbari et al., Curr Opin Immunol 15:627-633, 2003).
Autoimmune disease encompasses a wide spectrum of diseases that can affect many different organs and tissues within the body (see, e.g., Paul, W. E. (1999), Fundamental Immunology, Fourth Edition, Lippincott-Raven, New York.)
Current therapies for human autoimmune disease include glucocorticoids, cytotoxic agents, and recently developed biological therapeutics. In general, the management of human systemic autoimmune disease is empirical and unsatisfactory. For the most part, broadly immunosuppressive drugs, such as corticosteroids, are used in a wide variety of severe autoimmune and inflammatory disorders. In addition to corticosteroids, other immunosuppressive agents are used in management of the systemic autoimmune diseases. Cyclophosphamide is an alkylating agent that causes profound depletion of both T- and B-lymphocytes and impairment of cell-mediated immunity. Cyclosporine, tacrolimus, and mycophenolate mofetil are natural products with specific properties of T-lymphocyte suppression, and they have been used to treat systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and, to a limited extent, vasculitis and myositis. These drugs are associated with significant renal toxicity. Methotrexate is also used as a “second line” agent in RA, with the goal of reducing disease progression. It is also used in polymyositis and other connective-tissue diseases. Other approaches that have been tried include monoclonal antibodies intended to block the action of cytokines or to deplete lymphocytes (Fox, Am J Med 99:82-88, 1995). Treatments for multiple sclerosis (MS) include interferon and copolymer 1, which reduce relapse rate by 20-30% and only have a modest impact on disease progression. MS is also treated with immunosuppressive agents including methylprednisolone, other steroids, methotrexate, cladribine and cyclophosphamide. These immunosuppressive agents have minimal efficacy in treating MS. The introduction of the antibody Tysabri (natalizumab), an alpha 4-integrin antagonist, as treatment for MS has been overshadowed by incidences of progressive multifocal leucoencaphalopathy (PML) in patients receiving the therapy. Current therapy for RA utilizes agents that non-specifically suppress or modulate immune function such as methotrexate, sulfasalazine, hydroxychloroquine, leuflonamide, prednisone, as well as the recently developed TNFα antagonists etanercept and infliximab (Moreland et al., J Rheumatol 28: 1431-52, 2001). Etanercept and infliximab globally block TNFα, making patients more susceptible to death from sepsis, aggravation of chronic mycobacterial infections, and development of demyelinating events.
In the case of organ-specific autoimmunity, a number of different therapeutic approaches have been tried. Soluble protein antigens have been administered systemically to inhibit the subsequent immune response to that antigen. Such therapies include delivery of myelin basic protein, its dominant peptide, or a mixture of myelin proteins to animals with experimental autoimmune encephalomyelitis and humans with multiple sclerosis (Brocke et al., Nature 379: 343-6, 1996; Critchfield et al., Science 263: 1139-43, 1994; Weiner et al., Annu Rev Immunol 12: 809-37, 1994), administration of type II collagen or a mixture of collagen proteins to animals with collagen-induced arthritis and humans with rheumatoid arthritis (Gumanovskaya et al., Immunology 91: 466-73, 1999; McKown et al., Arthritis Rheum 42: 1204-8, 1999; Trentham et al., Science 261: 1727-30, 1993), delivery of insulin to animals and humans with autoimmune diabetes (Pozzilli and Gisella Cavallo, Diabetes Metab Res Rev 16: 306-7, 2000), and delivery of S-antigen to animals and humans with autoimmune uveitis (Nussenblatt et al., Am J Ophthalmol 123: 583-92, 1997). Another approach is the attempt to design rational therapeutic strategies for the systemic administration of a peptide antigen based on the specific interaction between the T-cell receptors and peptides bound to MHC molecules. One study using the peptide approach in an animal model of diabetes resulted in the development of antibody production to the peptide (Hurtenbach et al., J Exp Med 177:1499, 1993). Another approach is the administration of T cell receptor (TCR) peptide immunization (see e.g. Vandenbark et al., Nature 341:541, 1989). Still another approach is the induction of oral tolerance by ingestion of peptide or protein antigens (see e.g. Weiner, Immmunol Today 18:335, 1997).
Mucosal tolerance refers to the phenomenon of systemic tolerance to challenge with an antigen that has previously been administered via a mucosal route, usually oral, nasal or naso-respiratory, but also vaginal and rectal (Weiner et al., Annu Rev Immunol 12:809-837, 1994). Mucosal tolerance was discovered early in the 20th century in models of delayed-type and contact hypersensitivity reactions in guinea pigs, but the mechanisms of tolerance remained ill-defined until the era of modern immunology. The use of cell separation techniques, tests for production of cytokines and transgenic models in which antigen-specific T cells can be tracked in vivo have gradually elucidated mechanisms of mucosal tolerance (Garside and Mowat., Crit Rev Immunol 17:119-137, 1997). It has become evident that antigen administration via mucosal routes can result in distinct types of tolerance, depending on the route of administration and dose of antigen. For example, a high dose of oral antigen induces T-cell activation followed by deletion or anergy of responding T cells (Chen et al., Nature 376:177-180, 1995), analogous to parenteral administration of high-dose soluble antigen. This results in extinction of T cells specific to that antigen and unresponsiveness to subsequent antigen challenge, i.e. passive tolerance. In contrast, a low dose of oral antigen does not induce deletion or anergy but, when given repeatedly, induces a distinct type of immune response characterized by the appearance of regulatory-protective T cells, Treg cells, that secrete anti-inflammatory cytokines, i.e. active tolerance (von Herrath, Res Immunol. 148:541-554, 1997). These Treg cells usually belong to the class of CD4 (helper) T cells. Instillation of intact protein antigen onto the nasopharyngeal mucosa also induces Treg cells that are protective. In this case, both CD4 and CD8 T cells may be induced. Regulatory Treg cells induced after oral or intranasal antigen administration produce anti-inflammatory cytokines such as IL-4, IL-10 and TGF-β. To induce mucosal tolerance, antigen can also be given in the form of aerosol. Administration via these three routes, oral, intranasal and aerosol-inhalation, results in antigen uptake and presentation in different lymphoid compartments in each case. Accordingly, oral antigen is presented to T cells mostly in mesenteric lymph nodes and to some extent in Peyer's patches, intranasal antigen in deep cervical lymph nodes and inhaled antigen in mediastinal lymph nodes. Repeated exposure to antigen in each case is able to induce regulatory T cells, but the nature of these cells differs, depending on the route and form of antigen. While regulatory cells induced by oral antigen are CD4 T cells and express T cell receptors (TCR) consisting of αβ heterodimers, in the case of naso-respiratory antigen, the regulatory cells can also be CD8 T cells expressing a γδ heterodimer TCR (i.e. γδ T cells). Some of these cells may also have a CD8 receptor that is an αα homodimer instead of the conventional αβ-heterodimer TCR. A majority of cells that carry the CD8αα and γδ TCR reside in skin or mucosal tissues.
Over the past decades, there has been a significant increase in both the incidence and prevalence of allergic disease in western countries. Allergic rhinitis is the most common of these diseases, affecting 15-20% of the population. The allergic reaction is triggered by allergen-mediated cross-linking of specific IgE on the surface of mast cells and basophils, leading to release of histamine and other mediators, thus causing an acute allergic reaction, followed by a late-phase reaction characterized by an influx of eosinophils, neutrophils and Th2 cells producing IL-4, IL-5 and IL-13.
Specific immunotherapy (SIT) is recognized as an effective treatment of allergic rhinitis. Traditionally, SIT has been conducted by repeated subcutaneous administration of small amounts of specific allergen. Although this form of treatment can be an effective therapeutic option, concerns exist with the safety of this form of immunotherapy as well as with the difficulty of standardizing the allergen extract used as vaccine. Consequently, there is strong interest in the development of alternative and novel treatments against allergic diseases. One of the approaches is the use of mucosal vaccines (Widermann, Curr Drug Targets Inflamm Allergy 4, 577-583, 2005). Other alternatives are based on the use of allergen derivatives with reduced or no allergenicity as vaccines (Vrtala et al., Methods 32, 313-320, 2004). These include allergens obtained by protein engineering and synthetic peptides representing immunodominant T-cells epitopes of allergens. For example, Ole e1 has been identified as the most relevant allergen of olive pollen (Wheeler et al., Mol Immunol 27, 631-636, 1990).
Immune responses are currently altered by delivering polypeptides, alone or in combination with adjuvants (immunomodulating agents). For example, the hepatitis B virus vaccine contains recombinant hepatitis B virus surface antigen, a non-self antigen, formulated in aluminum hydroxide, which serves as an adjuvant. This vaccine induces an immune response against hepatitis B virus surface antigen to protect against infection. An alternative approach involves delivery of an attenuated, replication deficient, and/or non-pathogenic form of a virus or bacterium, each a non-self antigen, to elicit a host protective immune response against the pathogen. For example, the oral polio vaccine is composed of a live attenuated virus, a non-self antigen, which infects cells and replicates in the vaccinated individual to induce effective immunity against polio virus, a foreign or non-self antigen, without causing clinical disease. Alternatively, the inactivated polio vaccine contains an inactivated or ‘killed’ virus that is incapable of infecting or replicating and is administered subcutaneously to induce protective immunity against polio virus.
DNA therapies have been described for treatment of autoimmune diseases. Such DNA therapies include DNA molecules encoding the antigen-binding regions of the T cell receptor to alter levels of autoreactive T cells driving the autoimmune response (Waisman et al., Nat Med 2:899-905, 1996; U.S. Pat. No. 5,939,400). DNA molecules encoding autoantigens were attached to particles and delivered by gene gun to the skin to prevent MS and collagen induced arthritis. (WO 97/46253; Ramshaw et al., Immunol Cell Biol 75:409-413, 1997). DNA molecules encoding adhesion molecules, cytokines (e.g., TNFα), chemokines (e.g., C—C chemokines), and other immune molecules (e.g., Fas-ligand) have been used for treatment of autoimmune diseases in animal models (Youssef et al., J Clin Invest 106:361-371, 2000; Wildbaum et al., J Clin Invest 106:671-679, 2000; Wildbaum et al., J Immunol 165:5860-5866, 2000).
Methods for treating autoimmune disease by administering a nucleic acid encoding one or more autoantigens are described in WO 00/53019, WO 2003/045316, and WO 2004/047734. While these methods have been successful, further improvements are still needed.
Bacterial enterotoxins are used as immunostimulating adjuvants in vaccines for the prevention of infectious diseases. Cholera toxin (CT) and the closely related E. coli heat-labile toxin (LT) are perhaps the most powerful and best studied mucosal adjuvants in experimental use today (Rappuoli et al., Immunol Today 20:493-500), but when exploited in the clinic, their potential toxicity and association with cases of Bell's palsy (paralysis of the facial nerve) have led to their withdrawal from the market (Gluck et al., J Infect Dis 181: 1129-1132, 2000; Gluck et al., Vaccine 20 (Suppl. 1): S42-44, 2001; Mutsch et al., N Engl J Med. 350: 896-903, 2004). The bacterial enterotoxins CT and LT have proven to be effective immunoenhancers in experimental animals as well as in humans (Freytag et al., Curr Top Microbiol Immunol 236: 215-236, 1999). Structurally, these enterotoxins are AB5 complexes, and consist of one ADP-ribosyltransferase active A1 subunit and an A2 subunit that links the A1 to a pentamer of B subunits. The holotoxins bind to most mammalian cells via the B subunit (CTB), which specifically interacts with the GM1-ganglioside receptor in the cell membrane. Whereas the holotoxins have been found to enhance mucosal immune responses, conjugates between CTB and antigen have been used to specifically tolerize the immune system (Holmgren et al., Am J Trop Med Hyg 50: 42-54, 1994). Studies in mice have shown that CT and LT can accumulate in the olfactory nerve and bulb when given intranasally, a mechanism that is dependent on the ability of the B subunits of CT or LT to bind GM1-ganglioside receptors, present on all nucleated mammalian cells (Fujihashi et al., Vaccine 20: 2431-2438, 2002). Although less toxic mutants of CT and LT have been engineered with substantial adjuvant function, such molecules still carry a significant risk of causing adverse reactions (Giuliani et al., J Exp Med 187: 1123-1132, 1998; Yamamoto et al., J Exp Med 185: 1203-1210, 1997), especially when considering that the adjuvanticity of CT and LT appears to be a combination of the ADP-ribosyltransferase activity of the A subunit and the ability to bind ganglioside receptors on the target cells (Soriani et al., Microbiology 148: 667-676, 2002). These observations and others preclude the use of CT or LT holotoxins in vaccines for humans. On the other hand, recent observations have demonstrated that it is possible to retain adjuvant functions of these molecules with no toxicity or greatly reduced toxicity by introducing site-directed mutations in the gene coding for the A1 subunit. Examples of mutant molecules that have proven to be effective adjuvants are LTK63 and LTR72 (Giuliani et al., J Exp Med 187: 1123-1132, 1998), the former with no enzymatic activity and the latter with significantly reduced ADP-ribosylating ability. Notwithstanding this, the GM1-ganglioside receptor-dependent binding remains a problem in these mutants, and may therefore still cause nerve cell accumulation and neurotoxicity.
A better solution to this dilemma of efficacy versus toxicity is the CTA1-DD molecule that has proven to be a highly effective mucosal and systemic adjuvant (Ågren et al., J Immunol 158: 3936-3946, 1997; U.S. Pat. No. 5,917,026). This unique adjuvant is based on the enzymatically active A1-subunit of CT, combined with a dimer of an immunoglobulin-binding element from Staphylococcus aureus protein A. The molecule thereby avoids binding to all nucleated cells, which could result in unwanted reactions, and exploits fully the CTA1-enzyme in the holotoxin. Accordingly, all studies to date have found that CTA1-DD is nontoxic and has retained excellent immunoenhancing functions. When given systemically, CTA1-DD provides comparable adjuvant effect to that of intact CT, greatly augmenting both cellular and humoral immunity against specific immunogens coadministered with the adjuvant. It also functions as a mucosal adjuvant and should be safe, as it is devoid of the B subunit that is a prerequisite of CT holotoxin toxicity. CTA1-DD cannot bind to ganglioside receptors; rather, it targets B cells, limiting the CTA1-DD adjuvant to a restricted repertoire of cells that it can interact with. However, the adjuvant effect is not completely dependent on B cells, as been shown in strong induction of specific CD4 T cell immunity following intranasal immunizations using the CTA1-DD adjuvant in B-cell deficient mice (Eliasson et al., Vaccine 25: 1243-52, 2008, Akhiani et al., Scand J. Immunol 63: 97-105, 2006).
The adjuvant effect of CTA1-DD was absent in mutants CTA1-E112K-DD and CTA1-R7K-DD, which lack the ADP-ribosylating enzymatic activity (Lycke, Immunol Lett 97: 193-198, 2005).
WO 2009/078796 further describes immunomodulating complexes comprising the mutant CTA1-R7K-DD, and more specifically the immunomodulating complexes comprising CTA1-R7K-DD linked to the shared immunodominant collagen II peptide comprising amino acids 260-273 (CII260-273).
A conjugate of CTB and a peptide derived from bovine collagen II has been shown to be able to protect mice from developing collagen induced autoimmune ear disease as well as collagen-induced arthritis (Kim et al., Ann Otol Rhinol Laryngol 110: 646-654, 2001; Tarkowski et al., Arthritis Rheum 42: 1628-34, 1999). However, CTB may not be suited for human use due to its GM1-ganglioside-binding properties and potential neurotoxic effects, as discussed above.
The immunomodulating complexes of the present invention differ from the immounomodulating complexes comprising the mutant CTA1-R7K-DD according to WO 2009/078796 at least in that amino acid 187 has been changed from cysteine to alanine and, optionally, in that a lysine residue has further been inserted in the N-terminal of the mutant CTA1 subunit.
The inventors of the present invention have surprisingly found that further replacement of amino acid 187 cysteine by an alanine of the immunomodulating complexes comprising the mutant CTA1-R7K-DD according to WO 2009/078796, and in particular the immunomodulating complexes comprising CTA1-R7K-DD linked to the shared immunodominant collagen II peptide, provides a significantly improved therapeutic effect on arthritis, with significantly lower incidence and severity of arthritis in mice.
Without being bound by theory, the mechanism behind the surprising improvement in therapeutic effect of the CTA1-R7K/C187A-DD according to the present invention as compared to CTA1-R/K-DD according to WO 2009/078796 would seem explainable by the fact that the replacement of amino acid 187 cysteine by alanine abolishes the formation of dimers through disulfide bonds.
It was a priori unknown whether obtaining a therapeutic effect would be dependent on at least some or even a substantial degree of dimerisation of the resulting fusion-protein. Therefore, before the surprising findings of the present inventors, it was not predictable whether trying to avoid dimerisation would in fact be detrimental to therapeutic activity of the immunomodulating complexes, and whether making this amino acid replacement would result in a fusion-protein having at least as good therapeutic effects as the CTA1-R7K-DD construct.
Furthermore, the present inventors have surprisingly found that the insertion of a lysine residue in the N-terminal of the fusion protein drastically increases the expression and production of the fusion protein, K-CTA1-R7K/C187A-DD, without any loss with regard to the therapeutic efficacy of the protein due to misfolding, translocation or proteolytic degradation. Thus, it was hitherto unknown whether the insertion of a lysine residue in the N-terminal would be detrimental to the biological availability and therapeutic activity of the fusion protein (e.g. due to effects on folding, etc.), and whether the insertion of lysine in the N-terminal would result in a fusion protein with at least as good therapeutic effects as the CTA1-R7K-DD construct.